The maintenance of skeletal muscle mass, and the ability to increase mass in response to load, are crucial for whole body function and quality of life. The role of satellite cells, or muscle stem cells, in muscle mass regulation is equivocal. Effective muscle hypertrophy can occur independently of satellite cells; however, long- term muscle adaptation in the absence of satellite cells has not been explored. Preliminary data suggest that in the absence of satellite cells, muscle becomes fibrotic and hypertrophy plateaus. This raises the possibility that activated satellite cells play a paracrine role in regulating the muscle fiber niche. Excess extracellular matrix (ECM) production and muscle fibrosis, observed in muscular dystrophies and in aging, negatively impact muscle plasticity and function. Activated satellite cells may mediate skeletal muscle ECM production through modulation of transforming growth factor (TGF-) signaling. TGF- promotes increased production of ECM in muscle fibroblasts, and satellite cells may prevent TGF- downstream signaling, thereby influencing fibroblast activity and ECM production. It is hypothesized that in the absence of activated satellite cells, production of ECM by muscle fibroblasts increases via activation of TGF- signaling, resulting in muscle fibrosis and attenuated muscle growth and function. Further, we propose to test the novel hypothesis that muscle fibrosis, rather than a myonuclear domain ceiling, limits muscle hypertrophy in satellite cell-depleted muscle. Functional overload of plantaris muscle in two novel mouse strains will determine the role of activated satellite cells in modulating the extracellular environment to enable hypertrophy. The Pax7-DTA strain enables specific depletion of satellite cells, and the Pax7-N-WASp strain enables satellite cell activation and proliferation, but not fusion. Aim 1 will assess TGF- signaling activity, fibroblast proliferation, expression of ECM components, functional consequences of fibrosis, and the hypertrophic response in skeletal muscle following satellite cell depletion in the Pax7-DTA mouse in response to overload. Immunohistochemistry, fluorescent activated cell sorting, qRT-PCR, western blotting, biochemical assays and whole muscle function assessment will be performed. Increased fibroblast proliferation, expression of ECM components and depressed force production associated with reduced growth in satellite cell-depleted muscle will demonstrate a regulatory role for activated satellite cells in controlling the fiber extracellular environment. Aim 2 will utilize the Pax7-N-WASp mouse to determine the role of activated satellite cells in the hypertrophic response irrespective of their ability to contribute new nucleito growing fibers. Activated satellite cell regulation of fibroblast proliferation and ECM production will be assessed during muscle overload as in Aim 1. Restoration of hypertrophy in Pax7-N-WASp mice will demonstrate a novel role for activated satellite cells in adult skeletal muscle adaptability that may prove equally as important as their well-characterized role in muscle fiber regeneration.